Gene therapy based on recombinant adeno-associated virus (rAAV) vectors is showing great clinical promise. Previously, we showed that an intravenous rAAV injection could cause hepatocellular carcinoma (HCC) in newborn mice due to vector integration into and activation of a specific locus on chromosome 12 which we call the AAV-HCC locus in this proposal. Even a single integration event was sufficient to cause HCC in mice. Given that this locus is highly conserved and overexpressed in a subclass of human HCC, these mouse studies raise significant concerns about a possible risk of HCC induction in human gene therapy trials. In order to advance the promising field of liver-directed rAAV therapy, it is important to establish whether rAAV is likely to cause HCC in humans. In this proposal we systematically explore the risk of HCC caused by vector integration at the AAV-HCC locus, using three different animal models to establish the effects of clinically relevant risk factors, as well as vector design on HC induction. In addition, we will assess the risk conferred by random integration of rAAV gene therapy vectors in the liver. Our results will have a significant impact on the clinical practice o liver-directed gene therapy, not only for rAAV vectors, but also for any integrating vector, and may lead to new experimental models of human HCC.